Anti-apoptotic protein antibodies

ABSTRACT

Single-domain antibodies that bind pro-apoptotic proteins Bax and caspase-3 are identified and isolated. These single-domain antibodies may be used to modulate the activity of Bax and caspase-3, thereby modulating the symptoms and steps of oxidative stress and/or cell apoptosis, including Bax dimerization, mitochondrial permeabilization and the release of apoptotic proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/990,985,which is a national entry application claiming the benefit of PCTApplication No. PCT/CA2006/001451, which claims the benefit of U.S.Provisional Application No. 60/712,831, all of the above-referencedapplications being incorporated herein by reference.

SEQUENCE LISTING

Incorporated by reference herein is a sequence listing in electronicform, the file entitled 2012_(—)11_(—)23_sequence_listing.txt created onNov. 23, 2012 and having a size of 14 KB.

Field of the Invention

The invention relates to antibodies and fragments thereof which targetproteins with pro-apoptotic function, and methods for using suchantibodies.

BACKGROUND OF THE INVENTION

Programmed cell death or apoptosis is a physiological process essentialfor normal development and tissue homeostasis. Cell death mechanisms areprotective measures for organisms which ensure the removal ofunnecessary, damaged or potentially dangerous cells. However, anyderegulation or inappropriate induction of this process leads to theloss of healthy cells, causing diseases. In particular, cell death inpost-mitotic tissues such as the brain and heart in adult organismsresults in functional compromise, as is the case in Alzheimer's disease,Parkinson's disease and stroke. Cell death induced by oxidative stresshas been shown to be involved in the development of these pathologies.Although the exact mechanism of cell death induced by oxidative stressis still not known, mitochondria have been shown to play a central rolein this process. Mitochondrial events such as opening of thepermeability transition pores, mitochondrial membrane potential collapseand release of pro-apoptotic factors such as cytochrome c and/orapoptosis-inducing factors trigger the cascade of events leading toexecution of apoptosis.

Bax is a 24 kDa protein of the Bcl-2 family with pro-apoptotic function.It normally resides in cytosol and translocates to mitochondria uponinduction of apoptosis and it plays a key role in destabilizingmitochondria. Translocation of Bax to mitochondria followed by aconformational change (mitochondrial permeabilization) in associationwith Bid leads to the release of cytochrome c, apoptosis-inducing factorand caspase-9, a cysteine protease, which start the execution phase ofapoptosis. Bax has been implicated in neuronal cell death duringdevelopment and ischemia.

Caspase-3 is normally present in a dormant form. Once activated, itplays a role in the disintegration of various key proteins in the cell,including the activation of an endonuclease which fragments cell DNA.

Intrabodies to apoptotic proteins with inhibitory action would be usefulin the treatment of neurodegenerative disorders, in addition to beingvaluable tools for studying apoptosis. The efficacy of intrabodiescritically depends on their stability. In the reducing environment ofthe cytoplasm, intrabodies cannot form their stabilizing disulfidelinkage(s), so only those which are of sufficient stability can toleratethe absence of the disulfide linkage and be expressed in functionalform. Traditionally, single chain Fvs (single chain Fvs, or “scFvs”consist of an antibody heavy chain variable domain, V_(H), and a lightchain variable domain, V_(L), joined together by a linker) have beenused as intrabodies (Kontermann, R. E., 2004). More recently, thefeasibility of three types of single-domain antibodies (sdAbs), V_(L)s,V_(H)s and V_(H)Hs (V_(H)s derived from camelid heavy chain antibodies(Hamers-Casterman C. et al., 1993), as intrabodies has also beendemonstrated. While offering a comparable affinity, sdAbs have higherstability, solubility and expression level than scFvs and thus, are moreefficacious as intrabodies (Tanaka, T. et al., 2003;Aires da Silva, F.et al., 2004;Colby, D. W. et al., 2004) Intrabodies can be derived frommonoclonal antibodies or antibody display libraries, e.g., antibodyphage display libraries (Rondon, I. J. et al., 1997;Miller, T. W. etal., 2005).

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1. Superdex 200 size exclusion chromatogram showing the separationof a monomeric anti-Bax sdAb from a pentameric one (pentabody). In thesdAb pentabody, VTB represents the verotoxin B subunit, thepentamerization domain. The expected molecular weight of the pentabodyis 118 kDa.

FIG. 2. A. In vitro protection of mitochondria by anti-Bax V_(H)Hs.Measurement of ROS generation in isolated mitochondria (Mitoch).Isolated mitochondria were incubated with Bax either in the presence orabsence of different V_(H)Hs in a reaction buffer. ROS generation wasmeasured as described below. ROS generated by Bax alone was taken as100%. Different V_(H)Hs inhibited ROS production caused by Bax todifferent degrees with the best effect seen with Bax5-2 V_(H)H. Thestandard error shown as error bars was calculated using Microsoft Excelprogram and the data obtained from five separate experiments. P valueswere calculated using Statistica Application Program for Windows 95,where p values less than 0.05 were assumed to be significantlydifferent. Compared to the mitochondrial/Bax (M/B) the ROS decrease wassignificant in fractions containing Bax2 or Bax5-2 with p values under0.05, while fractions containing the irrelevant V_(H)HV_(H)H or Bax1 didnot show a statistically significant decrease in ROS generation(p-values above 0.05). B. Limited leakage of pro-apoptotic proteins frommitochondria in presence of anti-Bax V_(H)Hs. Cytochrome c retention andrelease from mitochondria was detected by Western blot in the pellet andsupernatant fractions (latter shown) of the reaction mixture containingisolated mitochondria incubated in the presence or absence of Bax withor without V_(H)H. Samples containing Bax5-2 V_(H)H and incubated withBax (lane 3) lead to a decrease in cytochrome c release in thesupernatant fraction than the control fraction containing mitochondriaand Bax only (lane 2) indicating a decrease in mitochondrial membranestabilization due to the presence of the anti-Bax intrabody. C.Quantification of band intensity for cytochrome c release was calculatedusing Chemilmanager V5.5 program based on the integrated density valuefor each band, indicating that the greatest amount of cytochrome c wasreleased by the fraction containing mitochondria and Bax alone (M/B).V_(H)H 5-2=Bax5-2

FIG. 3. A. Cloning and expression of V_(H)Hs in mammalian cells.Schematic diagram shows a V_(H)H expression construct in mammalianvectors in fusion with green fluorescent protein, GFP or red fluorescentprotein, RFP. The heptapeptide DPPVA™ links the C-terminus of the V_(H)Hto the N-terminus of the GFP or RFP. The parent vector alone with noV_(H)H, expresses the fluorescent proteins. For the expression of V_(H)Halone, the V_(H)H gene was cloned between Hind III and Not I restrictionendonuclease sites. ORF, open reading frame, denoting the maturetranslated product; P_(CMV), cytomegalovirus promoter; V_(H)H, V_(H)Hgene; GFP, GFP gene; RFP, RFP gene. B. Confirming the formation of astable cell line expressing anti-Bax intrabodies. Western blot analysisof expression of GFP-V_(H)H genes in mammalian cells: total proteinextract from cells transfected with GFP gene (˜30 kDa, lane 1) or eachof various V_(H)H-GFP genes (˜40 kDa, lane 2-4) were resolved onSDS-PAGE, transferred to nitrocellulose membrane and immunoblotted usinganti-GFP antibody as described herein. Numbers on the left side of thefigure show the locations of the molecular weight markers. C.Fluorescent microscopy showing the expression of Bax4-RFP in a stablecell line. SHSY-5Y cells were transected with V_(H)H expression vectors,creating three groups of unique stable cell lines expressing the sixanti-Bax V_(H)Hs in fusion with either RFP (shown here) or GFP or inabsence of a fusion protein. Geneticin was supplemented in order toselect only positively transfected cells. Here RFPs were used as markersfor V_(H)H expression, with positively transfected cells staining red.

FIG. 4 Monitoring nuclear morphology after oxidative stress. Nuclei ofcontrol cell lines and those expressing anti-Bax V_(H)Hs were monitoredusing Hoechst reagent to detect brightly stained, condensed nucleiindicative of apoptotic cells. All culture plates containing anti-BaxV_(H)H-expressing cells show very few apoptotic nuclei (Bax 3 and Bax5-2are shown) comparable to non-transfected/non-treated SHSY-5Y. Incontrast, control SHSY-5Y cell lines (non-transfected, RFP only or GFPonly) exposed to equal treatment have a greater number of apoptoticcondensing nuclei, in fact at this time the majority of these cells arecompletely dead and lifted off the culture plates and thus not capturedin the shown fields.

FIG. 5 Quantifying cell viability following oxidative stress. Cell linestransfected (tf) with anti-Bax V_(H)H-GFPs were treated (tr) withoxidative stress (100 μM H₂O₂ for 1 h). Control cells includednon-transfected/non-treated cells, ntf (ntr), non-transfected/treatedcells, ntf (tr), RFP-transfected/treated cells, RFP-tf (tr),GFP-transfected/treated cells, GFP-tf (tr) and PTH50-RFP-tf (tr) cells.After 24 h cultures were stained with Hoechst reagent (as described inFIG. 4). Healthy and apoptotic nuclei from three separate experimentswere counted using 6-10 fields/cell line/experiment, and the number ofhealthy cells was plotted as a percentage of all the cells counted asCell Viability. SHSY-5Y cells transfected with each of the six anti-BaxV_(H)Hs (fused with GFP or RFP) show strong resistance to apoptosiswhich was significantly different from all the control treated celllines (ntf(tr), GFP-tf(tr), RFP-tf(tr), PTHSO-tf(tr)) expressing pvalues less than 0.05.

FIG. 6. Detecting early phase apoptosis through Annexin V staining.Annexin V was used to monitor plasma membrane flipping resulting in agreen fluorescent outline of apoptotic cells. After the describedtreatment, cells lacking V_(H)H genes showed a greater proportion ofannexin V staining than the ones expressing the V_(H)Hs (Bax1 and Bax5-2V_(H)Hs are shown). Hoechst staining also shows a greater proportion ofhealthy nuclei in SHSY-5Y cells transfected with the anti-Bax V_(H)Hthan those transfected with RFP protein only.

FIG. 7. Post-oxidative stress cell division in cells expressing anti-BaxV_(H)Hs. Growth rates of the protected cells containing all six V_(H)Hs(Bax5-2-GFP-transfected cells are shown) remain unaffected aftertreatment with 200 μM H₂O₂ for 1 h. Cells were trypsinized and plated onfresh dishes 48 h after the H₂O₂ treatment and cell numbers were countedat day 1, 5 and 16 using trypan blue staining and a hemocytometer.

FIG. 8. Quantifying cell viability post increased oxidative stress.Twelve stable cell lines expressing the six anti-Bax V_(H)Hs in fusionwith GFP and RFP were all exposed to treatment with 200 μM H₂O₂ for 1 hand were monitored after 24 h. Cell viability was calculated showing asignificant survival rate in cells expressing Bax3 or Bax5-2 V_(H)Hs (nosignificant viability change was noted between each fusion proteinused). Non-transfected cells (ntf) or cells transfected only with RFP,GFP or an irrelevant V_(H)H (PTHSO), showed very poor survival rate(20-30%) when treated (tr) indicating that expression of anti-Bax V_(H)His necessary for resistance to oxidative stress. ntr, non-treated. Theseresults were further confirmed and shown to be statistically significantby calculating the p values. The cell viability for each of the anti-BaxV_(H)Hs was compared against each of the control treated cell lines,showing p values less than 0.05 and each of the control cell lines werealso shown to be statistically different from thenon-treated/transfected cells (p<0.05). ntr, non-treated.

FIG. 9. Anti-Bax V_(H)H expression causes mitochondrial stabilizationfollowing oxidative stress. Nuclear and mitochondrial staining wasperformed with Hoechst and JC-1 dyes, respectively, 24 h after theindicated treatment. Cells expressing all six anti-Bax V_(H)Hs in fusionwith GFP show healthy nuclei comparable to non-treated/non-transfectedcells (Bax1 and Bax5-2 V_(H)Hs are shown). Mitochondrial membranedestabilization was monitored using JC-1. Healthy cells containingmitochondria with intact membrane potential show red fluorescence as innon-treated cells and cells containing V_(H)H (not done withRFP-transfected cells since the red would show from both the RFP andhealthy mitochondria).

FIG. 10 Mitochondrial membrane potential is protected in the presence ofanti-Bax V_(H)Hs. The mitochondrial membrane potential was measuredusing a cationic dye which accumulates in healthy mitochondria and canbe detected quantitatively using a fluorescence plate reader.Non-treated SHSY-5Y cells, ntf (ntr), with healthy mitochondriaexpressed high fluorescence/pg protein readings similar to the cellsexpressing Bax5-2 V_(H)H and treated with 100 μM H₂O₂/1 h. Conversely,non-transfected SHSY-5Y, ntf (tr), exposed to the same treatment showeda significant decrease in fluorescence indicative of destabilizedmitochondrial membrane potentials.

FIG. 11. Anti-Bax V_(H)Hs prevent lipid peroxidation after oxidativestress. Lipid peroxidation was monitored in cells treated with 100 μMH₂O₂ for 1 h. Cells expressing all six anti-Bax V_(H)Hs (Bax 3 andBax5-2 are shown) showed a drastic decrease of lipid peroxidationcompared to the control cells (non-transfected/treated, ntf (tr)) whichwere taken as 100% peroxidation. Lipid peroxidation levels of thetransfected cells (tf) were similar to that ofnon-transfected/non-treated (ntf (ntr)) cells. Compared to the ntf (ntr)cells, the ntf (tr) and PTH50-transfected (tr) cells the lipidperoxidation percentages in these latter cell lines were shown to bestatistically different (p<0.05). Furthermore, cell lines expressinganti-Bax V_(H)Hs showed lipid peroxidation which was statisticallydifferent from the two control treated cell (tr) (p<0.05) but similar tothe ntf (ntr) cells (p>0.05).

FIG. 12. Caspase 3/7 activation was prevented by anti-Bax intrabodies. Ahigh throughput screening assay was used to quantify the activation ofCaspase 3/7 in non-transfected (ntf) control (non-treated, ntr, ortreated, tr) cells as well as those expressing Bax5-2 V_(H)Hs. Afluorescence plate reader was used to detect the fluorescence produceddue to high levels of active caspases. Oxidative stress was inducedusing 100 μM H₂O₂ for 1 h and readings were taken after 6 h. Cellsexpressing Bax5-2 V_(H)H produced low levels of fluorescence comparableto control cell line without any treatment (ntf (ntr)), indicating adecrease in caspase activation due to protection of mitochodria by theanti-Bax intrabody. In contrast, non-transfected, treated cells (ntf(tr)) produced significantly high levels of fluorescence, with p valuesless than 0.05 when compared to both ntf (ntr) and Bax5-2 expressingcells, indicating strong caspase activation in these control cell lines.

FIG. 13. Protection by V_(H)Hs against oxidative stress is not affectedby the presence of the fusion fluorescent proteins. Stable cell linesexpressing V_(H)Hs (Bax1, Bax2, Bax3, Bax5-1 and Bax5-2) without theGFP/RFP fusion proteins were also established and tested for survivalunder oxidative stress conditions. Apoptosis was decreased in these celllines which exhibited cell viability rates significantly higher than allthe treated control cell lines (ntr, GFP-tf(tr), RFP-tf(tr),PTHSO-tf(tr)), as the p values for all the anti-Bax V_(H)H expressingcells were <0.05 when compared to each of the aforementioned treatedcontrol cells.

FIG. 14. Amino acid sequences of the anti-Bax V_(H)Hs. CDR1, CDR2 andCDR3 appear sequentially in bold text (SEQ ID NOS 1-6 are disclosedrespectively in order of appearance). Dots represent sequence identitywith Bax2 V_(H)H, and dashes are included for sequence alignment. Kabatnumbering system is used (Kabat et al., 1991).

FIG. 15. Binding analysis of the anti-Bax V_(H)Hs. (A) Binding ofV_(H)H-displayed phages to immobilized Bax by ELISA. None of theV_(H)H-phages bound to BSA, and the phage alone showed a backgroundbinding to Bax. Definitive conclusions with respect to the relativeaffinity of the V_(H)Hs for Bax cannot be drawn, because the amount ofV_(H)H-phage added during the binding step is not known. (B) Binding bysurface plasmon resonance showing sensorgram overlays for the binding of3.4 μM, 6.7 μM, 10 μM, 13 μM, 17 μM, 20 μM, 23 μM and 27 μM Bax2 V_(H)Hto immobilized Bax.

FIG. 16. Binding of anti-caspase-3 V_(H)H-displayed phages toimmobilized caspase-3 by ELISA. None of the V_(H)H-phages bound to BSA,and the phage alone showed a background binding to Bax.

FIG. 17. Amino acid sequences of Casp1 (A) (SEQ ID NO: 7) and Casp2 (B)(SEQ ID NO: 8) V_(H)Hs. CDR1, CDR2 and CDR3 appear sequentially,underlined and in bold text. CDR designations are based on Kabatnumbering system (Kabat et al., 1991).

FIG. 18. Effects of Casp1 and Casp2 sdAbs on active Caspase 3 isolatedfrom SHSY-5Y cells induced by treatment with 50 μM H₂O₂. Control isactive caspase 3 without sdAbs added and Casp1 and Casp2 are taken aspercentages relative to the control.

SUMMARY OF THE INVENTION

A first object of this invention is to identify and isolatesingle-domain antibodies or fragments thereof which bind to apoptoticproteins such as Bax and caspase-3.

A second object of this invention is to provide a method for modulatingapoptosis or its effects through the use of single-domain antibodieswhich bind to apoptotic proteins such as Bax and caspase-3.

A further object of this invention is to provide a method for treatingdiseases or conditions, where disease symptoms are caused by undesirablecell apoptosis or oxidative stress, or where targeted cell apoptosis isdesired.

There is disclosed herein the identification, cloning and functionalcharacterization of several Bax-specific and caspase-3-specific singledomain antibodies (sdAbs). These minimal size antibody fragments, whichwere isolated from a llama V_(H)H phage display library by panning,inhibit anti-Bax or anti-caspase-3 function in in vitro assays.Importantly, as intrabodies, these sdAbs, which were stably expressed inmammalian cells, were nontoxic to their host cells and rendered themhighly resistant to oxidative-stress-induced apoptosis. Theseintrabodies are useful drugs on their own as well as a means foridentifying small compound drugs for degenerative diseases involvingoxidative-stress-induced apoptosis. The single domain antibodies andfragments may be used in the context of gene therapy or bound to aminoacid sequences that allow the sdAbs to be brought into cells.

A first aspect of the invention provides for a single-domain antibodyhaving a binding affinity for an apoptotic protein. In particular, suchsingle-domain antibody may bind Bax or caspase-3, and such binding mayinhibit or activate Bax or inhibit or promote the activation ofcaspase-3.

The single-domain antibody may have an amino acid sequence thatcomprises at least one of SEQ. ID NO.: 1, SEQ ID NO.:2, SEQ ID NO.3, SEQID NO.4, SEQ ID NO.5, SEQ ID NO.: 6, SEQ ID NO.: 7 and/or SEQ ID NO.: 8,or a variant or fragment thereof.

A second aspect of the invention provides for a multimer, and preferablya pentamer, comprising at least two single-domain antibodies having abinding affinity for an apoptotic protein, such as Bax or caspase-3.

A third aspect of the invention provides for a vector comprising anucleic acid sequence encoding a single-domain antibody that binds anapoptotic protein, and a cell, preferably a human cell, that comprisesthe vector.

A further aspect of the invention provides for a method for modulatingthe symptoms of apoptosis in a cell, comprising the steps of exposingthe cell to at least one single-domain antibody having a bindingaffinity for Bax; and allowing binding of the at least one single-domainantibody to Bax.

A further aspect of the invention provides for a method for modulatingmitochondrial permeabilization in a cell, comprising the steps ofexposing the cell to at least one single-domain antibody having abinding affinity for Bax; and allowing binding of the at least onesingle-domain antibody to Bax.

A further aspect of the invention provides for a method of modulatingBax-Bax dimerization in a cell comprising the steps of exposing the cellto at least one single-domain antibody having a binding affinity forBax; and allowing binding of the at least one single-domain antibody toBax.

A further aspect of the invention provides for a method for modulatingthe effects of oxidative stress in a cell comprising the steps ofexposing the cell to at least one single-domain antibody having abinding affinity for Bax; and allowing binding of the at least onesingle-domain antibody to Bax.

A further aspect of the invention provides for a method for modulatingthe production of reactive oxygen species in a cell comprising the stepsof exposing the cell to at least one single-domain antibody having abinding affinity for Bax; and allowing binding of the at least onesingle-domain antibody to Bax.

A further aspect of the invention provides for a method for modulatinglipid peroxidation in a cell comprising the steps of exposing the cellto at least one single-domain antibody having a binding affinity forBax; and allowing binding of the at least one single-domain antibody toBax.

A further aspect of the invention provides for a method for modulatingthe release of apoptotic proteins within a cell, comprising the stepsexposing the cell to at least one single-domain antibody having abinding affinity for Bax; and allowing binding of the at least onesingle-domain antibody to Bax.

A further aspect of the invention provides for a method for treating adisease or condition involving cell death, comprising the steps ofexposing the cell to at least one single-domain antibody having abinding affinity for Bax; and allowing binding of the at least onesingle-domain antibody to Bax.

A further aspect of the invention provides for a method for treating adisease or condition involving cell death comprising the steps ofexposing the cell to at least one single-domain antibody having abinding affinity for caspase-3; and allowing binding of the at least onesingle-domain antibody to caspase-3.

A further aspect of the invention provides for a method for treatingcancer through induction of apoptosis in cancer cells, comprising thesteps of exposing the cancer cells to at least one single-domainantibody having a binding affinity for Bax or caspase-3 and allowingbinding of the at least one single-domain antibody to Bax or caspase-3.

A further aspect of the invention provides for the use of a firstsingle-domain antibody or antibody fragment having a binding affinityfor Bax or caspase-3 for identifying a second single-domain antibody orantibody fragment having a binding affinity for an apoptotic protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a variety of potential antibodies andantibody-derived fragments, including single domain fragments. Asreferred to herein, a single-domain fragment is a protein fragmenthaving only one domain, the domain being preferably a variable domainderived from the immunoglobulin superfamily. It will be understood thatsingle domain fragments may be produced by translation of all or part ofa nucleic acid sequence or by other methods, including de novo chemicalsynthesis, and fragmentation of larger proteins, such as proteasetreatment of immunoglobulins. The fragment could also be an Igsuperfamily variable-like domain such as the type III domain offibronectin and the cytotoxic T lymphocyte associated antigen-4 (CTLA-4)extracellular domain.

The single-domain antibodies identified herein were obtained byscreening a naïve llama V_(H)H phage display library. Several sdAbs wereisolated and characterized, and found to have binding affinities forapoptotic proteins, including Bax and caspase-3. Such binding affinitiesmay be used to inhibit the activity of these apoptotic proteins, oralternatively to activate the proteins.

The single-domain antibodies identified include sdAbs having amino acidsequences SEQ ID NO.: 1 through SEQ ID NO.: 8 shown in the attachedsequence listing. These antibody fragments were found to have apoptoticprotein binding affinity. It is expected that variants or fragments ofthese sequences having apoptotic protein binding affinity would also beuseful. The corresponding nucleic acid sequences are shown as SEQ IDNO.: 10 through SEQ ID NO.: 17. Mutants, variants, homologs or fragmentsof these nucleic acid sequences encoding sdAbs with apoptotic proteinbinding affinity will also be useful.

Single domain fragments may be modified to add additional moieties insome cases. Examples of additional moieties include polypeptides suchantibody domains, marker proteins or signal proteins. Examples ofantibody domains include, but are not restricted to, Immunoglubulin (Ig)light chain variable domains (V_(L)), Ig heavy chain variable domains(V_(H)), camelid (camels and llamas) heavy chain antibody variabledomains (V_(H)H), nurse shark and wobbegong shark Ig new antigenreceptor (IgNAR) variable domains (V_(H)), T cells receptor variabledomains. Examples of marker proteins include red or green fluorescentprotein, or radioisotopes. Examples of signal proteins includedmitochondrial or nuclear transport signal proteins. Another possibilityis the addition of leader sequences to the nucleic acid sequencesencoding the sdAbs—these could determine cellular localization of thesdAbs.

The single-domain antibodies or fragments of the present invention maybe incorporated into viral or plasmid vectors. These vectors can then beincorporated into cells, including human cells.

The non-toxic, Bax-specific and caspase-3-specific V_(H)H intrabodies ofthe present invention phenotypically transform their host neuronal cellsinto cells which are resistant to oxidative-stress-induced apoptosis.This opens new opportunities for treating neurodegenerative diseaseswhich involve cell death induced by oxidative stress and Bax activation.In particular, the sdAbs and fragments of the present invention may beused to modulate the symptoms of cell apoptosis. Exposure of these sdAbsand fragments to cells (typically by the expression of the sdAbs withinthe cells) can promote or prevent apoptosis through binding of the sdAbswith apoptotic proteins such as Bax and caspase-3. For example, bindingof sdAbs to Bax can prevent or promote mitochondrial permeabilization,as Bax is thought to play a significant role in this process. Thus, theinhibition of Bax can prevent mitochondrial permeabilization, while theactivation of Bax can promote this process. Closely related tomitochondrial permeabilization is the release of apoptotic proteins suchas cytochrome c, apoptosis inducing factor, and caspase-9, andaccordingly Bax-binding sdAbs may be used to modulate the release ofapoptotic proteins.

Other examples of uses for Bax-binding sdAbs include modulating theeffects of oxidative stress in cells, modulating the production ofreactive oxygen species in cells, and modulating lipid peroxidation incells. Oxidative stress may lead to cell apoptosis, and accordingly theuse of Bax or caspase-3 binding sdAbs to promote or prevent apoptosisallows for modulation of the effects of oxidative stress. The productionof reactive oxygen species and the peroxidation of cell lipids areexamples of effects caused by oxidative stress in the cell, andaccordingly these can be modulated through the use of sdAbs with bindingaffinities for apoptotic proteins.

Similarly, the sdAbs of the present invention may be used to promote orprevent the dimerization of Bax. Where, for example, sdAbs bind to Baxat its dimerization site or in such a way that Bax dimerization is notpossible, the apoptotic processes initiated by the activated Bax dimercannot take place. By contrast, where sdAbs bind to Bax at a site thatdoes not block Bax-Bax dimerization, this dimerization may be promotedby, for example, the use of multimerized sdAbs as discussed below.

Several research groups are working towards utilizing intrabodies astherapeutic agents in various diseases (Miller, T. W. et al., 2005).Other than the direct use of these intrabodies, these sdAbs could beused as biochemical tools to fish out specific and non-toxic inhibitorsof

Bax from pharmacophore libraries. Furthermore, fluorescence orradio-labeled anti-Bax sdAbs and the oxidative—stress resistant celllines would be a valuable research tools to elucidate the mechanism ofmitochondrial permeabilization and apoptosis in general.

The current anti-Bax V_(H)Hs and single domain fragments can be used inthe diagnosis and therapy of several diseases, especially thoseinvolving cell death, including neurodegenerative diseases,cardiovascular diseases, stroke, AIDS and cancer.

Anti-caspase fragments can be used in the treatment and amelioration ofa variety of diseases and disorders, either to induce or to inhibitapoptosis. For example, anti-caspase single domain fragments capable ofinhibiting caspase-3 activity or inhibiting activation of caspase-3 canbe used in blocking cell death in Alzheimer's disease, Parkinson'sdisease, AIDS and stroke. Conversely, anti-caspase or anti-Bax singledomain fragments capable of activating caspase-3 may also be used asanticancer agents to induce apoptosis in cancer cells.

Delivery of the anti-caspase and anti-Bax antibodies and antibodyfragments to cells may be accomplished by various methods. In thecontext of gene therapy, nucleic acid sequences encoding the antibodiesmay be delivered into cells as viral vectors, such as adenovirus,vaccinia virus or adeno-associated virus. For example, a protein such asan antibody or antibody fragment having specificity for a particularcell surface molecule may be attached to the surface of the virus,allowing the virus to target specific cells. Further, the virus may beengineered to contain nucleic acid sequences, such as promoters, whichallow the virus to function in only particular cells, such as cancercells.

Another option is the delivery of single-domain antibodies in the formof immunoliposomes. Such liposomes may contain single-domain antibodiesor fragments (as genes or as proteins), and may be designed tospecifically target the DNA-lipid complex of the target cells.

Single-domain antibodies may also be delivered to cells such as cancercells in nucleic acid form through a bifunctional protein. For example,the bifunctional protein may include both an antibody specific to acancer cell and a nucleic acid binding protein such as human protamine.The binding protein would attach to the sdAb gene and the antibody wouldallow specific cells to be targeted.

Alternatively, treatment for these diseases may be accomplished throughdelivery to cells in a protein form. This may be accomplished by fusingthe sdAb to a membrane translocating sequence (MTS) or proteintransduction domain (PTD) to allow for transportation across the plasmamembrane. Another option is to fuse the sdAb to an internalizing protein(eg. internatlizing antibody or antibody fragment) which allows the sdAbto be internalized by the cell.

In cases where it is desirable for the single-domain antibodies to crossthe blood-brain barrier, the sdAb may be fused to a polypeptide capableof crossing this barrier, or the nucleic acid sequence encoding the sdAbmay be part of a viral vector which is capable of crossing the barrier.Additional means (as discussed above) for delivering the sdAb to braincells once it has crossed the blood-brain barrier would still berequired.

In a therapeutic setting, the V_(H)Hs can also exert their effect bymodulating the action of Bax by functioning as shuttles, taking Bax todesired cell compartments, e.g., nucleus. This is done by attachingspecific signal sequences to V_(H)Hs through genetic engineering ormolecular biology techniques.

In some instances it will be desirable to use conjugates or fusionproteins comprising single domain binders and a domain allowing homo orhetero-multimerization. In particular, as discussed below, the formationof multimers, and in particular pentamers, of single domain intrabodiesmay increase the binding affinity of the sdAbs.

One possible application of a multimerized sdAb would be that more thanone bound apoptotic protein could be brought together. This may beuseful in the case of apoptotic proteins such as Bax and caspase-3 whichdimerize or multimerize in order to take an active form through, forexample, cleaving of sulphide bonds as in the case of caspase-3.

Accordingly, if two or more inactive caspase-3 molecules are broughttogether by a sdAb pentamer or other multimer, they can cleave eachother and thus be activated.

Identification of anti-Bax V_(H)Hs

Anti-apoptotic single-domain intrabodies were identified by screening anaïve llama V_(H)H phage display library (Tanha, J. et al., 2002e)against Bax. Screening of 38 colonies from the second and the thirdrounds of panning gave six different V_(H)H sequences, namely, Bax1,Bax2, Bax3, Bax4, Bax5-1 and Bax5-2, occurring at frequencies of 9, 24,2, 1, 1 and 1, respectively (FIG. 14). All six V_(H)Hs bound strongly toBax but not to control BSA antigen in phage ELISAs (FIG. 15A). V_(H)Hswere expressed as fusion protein with C C-terminal c-Myc-His₅ tag (SEQID NO: 9) in E. coli and purified to homogeneity for subsequentfunctional studies. Bax2 was chosen to provide an example for confirmingthe ELISA binding data by surface plasmon resonance (SPR) because of itsavailablity at desirable quantities. The V_(H)H was specific to Bax, asit did not bind to a control Fab, with equilibrium dissociation constant(K_(D)) of 40 μM (FIG. 15B). Binders obtained previously from the naïveV_(H)H phage display library (Tanha, J. et al., 2002d) have had K_(D)sin the low micromolar range with K_(D)s of a few micromolar at best(Yau, K. Y. et al., 2003;Yau, K. Y. et al., 2005).

Functional Characterization of V_(H)Hs In Vitro: Inhibition of BaxActivity in Isolated Mitochondria

The ability of the six V_(H)Hs to inhibit Bax was tested by monitoringBax-induced ROS generation from isolated mitochondria, which asmentioned above correlates with mitochondrial destabilization (Nomura,K. et al., 2000). We hypothesized that if the anti-Bax V_(H)Hs areinhibitory towards Bax, pre-incubation of isolated mitochondria withanti-Bax V_(H)Hs, followed by the addition of Bax should prevent Baxfrom permealizing the mitochondria and lead to a reduced ROS releasefrom mitochondria into the solution. Indeed, for all the V_(H)Hs (Bax1,Bax2, Bax3, Bax4, Bax5-1 and Bax5-2) tested, we observed a significantdecrease in

ROS release from mitochondria incubated with V_(H)Hs and Bax compared tothe fractions of mitochondria incubated with Bax alone or with Bax andan irrelevant V_(H)H (p<0.05) (FIG. 2A). Specifically, Bax3 and Bax5-2V_(H)Hs showed greatest potential as Bax inhibitors decreasing ROSproduction from mitochondria by approximately 55% and 90%, respectively.V_(H)Hs can inhibit the Bax function by binding to Bax at several sites:on the Bid-binding site, on the transmembrane domain and/or at the siteinvolved in Bax-Bax dimerization and activation. The variability ofinhibition by different sdAbs suggests that these sdAbs might beblocking different sites on Bax. Specifically, Bax5-2 is likely bindinga site involved in Bax function as it has the maximum inhibitory effect.

Permeability of the mitochondria was also monitored through detection ofcytochrome c release by Western blot. Cytochrome c is released from theinner mitochondrial space into the solution when this organelle isdestabilized (Adhihetty, P. J. et al., 2003). Thus, stable and healthymitochondria are expected to show strong retention of cytochrome c. Asin the previous ROS assay, isolated mitochondria in solution werepre-incubated with V_(H)Hs followed by the addition of Bax (mitochondriaalone and in presence of recombinant Bax protein only were used ascontrols). When incubated with Bax, significantly higher cytochrome crelease was seen in the supernatant fraction of the mitochondriaincubated with recombinant Bax protein alone compared to those incubatedwith V_(H)Hs and Bax (FIG. 2B), these findings were further confirmed bycalculating the percent integrated density value of each band intensity(FIG. 2C). Equal protein sample loading was confirmed in both instancesby Ponseau S staining of the blots before incubation with blockingsolution (data not shown). Conversely, mitochondria preincubated withV_(H)Hs showed significantly more cytochrome c in its pellet fraction(representing intact mitochondrial membrane) than the ones with nopreincubation, demonstrating that the V_(H)Hs decreased the permeabilityof mitochondria initiated by Bax (data not shown).

Functional Characterization of Anti-Bax V_(H)Hs In-Situ: Inhibition ofApoptosis by V_(H)Hs when Expressed as Intrabodies in Mammalian Cells

The assays performed on isolated mitochondria clearly indicated that theV_(H)Hs can bind and prevent Bax activity in solution and in isolatedmitochondria. We further studied the effects of the V_(H)Hs asintrabodies inside intact cells. SHSY-5Y cells were transfected with allsix V_(H)H genes in fusion with RFP and GFP (FIG. 3A) and 12 stable celllines, each expressing a unique V_(H)H fusion protein, were obtained. Inaddition, stable control cell lines containing genes for an irrelevantV_(H)H (PTHSO, a parathyroid-hormone binding V_(H)H) or fluorescentproteins alone were established. Expression of the V_(H)Hs from bothtransient and stable transfections was confirmed by Western blotanalysis of cell lysates using anti-GFP antibody (FIG. 3B). In lane 1,cells containing GFP produce a band just under 30 kDa, consistent withthe GFP molecular weight, 27 kDa (Battistutta, R. et al., 2000), whilecells expressing each of the six anti-Bax V_(H)H-GFP fusion proteinsproduce a band at approximately 40 kDa, very close to the theoreticalvalue, 41 kDa (Bax5-2, Bax3 and Bax2 are shown). Fluorescence microscopywas also used to “visualize” expression of V_(H)H-RFP or V_(H)H-GFPfusion proteins in cells (FIG. 3C, Bax4-RFP is shown).

To assess the protective capabilities of the six Bax-specific V_(H)Hs incontext of intrabodies, we again monitored the resistance of cells toapoptosis under oxidative stress. As previously discussed, oxidativestress due to mitochondrial ROS elevation, has been linked to theactivation of Bax and ultimate destabilization of the mitochondrialeading to apoptosis (Susin, S. A. et al., 1999;Adhihetty, P. J. et al.,2003). Thus, we hypothesized that if the V_(H)H intrabodies block Baxactivity during oxidative stress, apoptosis would be prevented. Inprevious studies it was shown that exposure of SHSY-5Y cells to 100 μMH₂O₂ for 1 h results in a significant increase in the rate of apoptosis(Somayajulu, M. et al., 2005). By implementing this condition to thestable cell lines containing either a V_(H)H gene or a control gene wemonitored the cells 24 h after H₂O₂ treatment for apoptotic features. Tothis end, the cells were stained with Hoechst reagent where brightlystained and condensing nuclei would be indicative of apoptotic cells(FIG. 4).

Untreated SHSY-5Y cells not expressing any V_(H)H were used as apositive control with approximately 96% cell viability (FIG. 5). Whenthe three negative control cell lines (non-transfected, transfected withGFP or RFP only or transfected with PTHSO) were exposed to 100 μM H₂O₂,a significant number of cells underwent apoptosis 24 h after thetreatment as indicated by brightly stained condensed nuclei. Thus, thenumber of non-apoptotic healthy cells was reduced to approximately 50%(FIG. 5). Interestingly, the cells containing any of the six anti-BaxV_(H)H intrabodies in fusion with GFP or RFP showed very good resistanceto the similar treatment with 87-93% viabilities and variedsignificantly from all the control treated cell lines (p<0.05). Thesevalues are very close to the viability values for the untreated,nontransfected SHSY-5Y cells (96%), demonstrating the effectiveness ofthe V_(H)H intrabodies in preventing apoptosis.

Annexin V in parallel with Hoechst staining (FIG. 6) was used to monitorplasma membrane flipping (another indicator of the early phase ofapoptosis) and nuclear condensation respectively. These assays furtherconfirmed the above finding that apoptosis was inhibited in the cellsexpressing anti-Bax V_(H)Hs. Moreover, the expression of V_(H)Hs wasnon-toxic to their host cells as growth and proliferation was nothindered. The V_(H)H-containing cells that survived the oxidative stresswere viable and fully functional after several days following H₂O₂treatment (FIG. 7) as their growth rate was similar to those without anyoxidative stress.

Intrabodies Prevent Mitochondrial Membrane Potential Collapse FollowingOxidative Stress and Render the Host Cells Resistance to Apoptosis atHigher Level of Oxidative Stress

To further assess the degree of potency of the V_(H)H intrabodies inpreventing apoptosis, we increased the stress conditions. When the H₂O₂(100 μM) exposure time was increased to 3 h, cells expressing V_(H)Hsshowed almost identical survival rates as when exposed for 1 h (data notshown). When treated with 200 μM H₂O₂ for 1 h, control cell lines(containing no or the irrelevant V_(H)H) showed a very high degree ofapoptosis and poor survival as measured by Hoechst staining and trypanblue exclusion assay. Conversely, almost all cell lines contain each ofthe V_(H)Hs showed significant survival, with the most promising beingthe cells containing Bax1, Bax2, Bax3, Bax4, BaxS-1 and BaxS-2 V_(H)Hs(FIG. 8). As before, the cell viabilities of the anti-Bax V_(H)Hexpressing cells were shown to be statistically higher than all thecontrol treated cell lines (p<0.05). Importantly, these results are inagreement to the ROS data (FIG. 2A) obtained using V_(H)Hs in presenceof isolated mitochondria. In addition, JC-1 staining of mitochondrialmembrane potential after the 200 μM H₂O₂/1 h treatment (FIG. 9) showedstable mitochondrial potential in cells expressing the anti-Bax V_(H)Hscomparable to the non-transfected, non-treated control SHSY-5Y. Theseresults were further quantified using a newly developed mitochondrialmembrane potential assay called MitoCasp assay. In this assay, a cellpermeable cationic dye when exposed to cell fractions is accumulated inhealthy mitochondria and exhibits a strong fluorescence signal (in thered), which can be measured using a fluorescence plate reader. Collapseof mitochondrial membrane potential leads to a decrease in thefluorescence. Results shown in FIG. 10 indicated that there was aconsiderable decrease in the fluorescence in the non-transfected SHSY-5Ycells after 100 μM H₂O₂/1 h treatment, compared to the non-treatedcontrol cells and the treated Bax5-2 expressing cells (p<0.05).Conversely, cells expressing Bax5-2 V_(H)H produced strong fluorescencecomparable to control non-transfected/non-treated SHSY-5Y cell (p>0.05).This data further confirms the ability of these intrabodies to protectthe mitochondria from Bax-mediated permealization in the presence ofoxidative stress (FIG. 10).

In addition, we also monitored lipid peroxidation, another indicator ofoxidative stress. When cells are exposed to higher ROS levels, mostcommonly due to mitochondrial damage, lipid deterioration is observed(Sunderman, F. W., Jr. et al., 1985). Lipid peroxidation was assessedfor cells expressing anti-Bax V_(H)Hs as well as non-transfected cells(with and without treatment), 24 h after exposure to 100 μM H₂O₂ for 1h. We observed a significant decrease in lipid peroxidation in cellsexpressing anti-Bax V_(H)Hs, compared to non-transfected/treated SHSY-5Ycells (FIG. 11; Bax3 and Bax5-2 are shown) (p<0.05). This indicates thatthe V_(H)Hs are able to block mitochondrial permeabilization by Bax,thus, limiting the leakage of various apoptotic inducing factors andultimately preventing cell death.

Activation of executioner caspases 3/7 was also monitored in V_(H)Htransfected cells using a high throughput screen for Caspase 3/7 assaykit measuring the activity of these proteases as an increase influorescence. Specifically, this kit utilized a quenched (z-DEVD)₂-R110peptide which is cleaved by active caspases 3/7 to release R110 free dyefrom the quenching caspase substrate DEVD. In this way the increase influorescence is indicative of capsase 3/7 activation in vivo. For thisassay, oxidative stress was induced in control non-transfected(non-treated or treated) cells and Bax5-2 V_(H)H-expressing cells (100μM H₂O₂/1 h) and caspase activation was measured after 6 h. We observeda significant increase in fluorescence indicative of strong caspaseactivation in control non-transfected/treated SHSY-5Y cells which wassignificantly lower in non-transfected/ non-treated and Bax5-2expressing cells (p<0.05) (FIG. 12).

Presence of GFP or RFP as Fusion Proteins does not Alter theAnti-Apoptotic Activities of Anti-Bax Intrabodies

To further show that anti-apoptotic activities of the anti-Bax V_(H)Hsare independent of their fusion context, cell lines of V_(H)Hintrabodies without fusion to GFP or RFP were also established. Thesecells were also monitored for their ability to resist oxidative stressinduced apoptosis by treatment with 200 μM H₂O₂ for 1 h. Apoptosis wasmonitored after 24 h using Hoechst staining to detect apoptotic nuclei.As shown in FIG. 13, cells expressing the “un-fused” anti-Bax V_(H)Hshave cell viability rates which were significantly higher than all thetreated control cell lines (p<0.05), indicating that the cellstransfected without the fluorescent marker protein have comparable cellsurvival rates to their respective cells with fused V_(H)H. Theseresults clearly indicate that inhibition of apoptosis was solely due theV_(H)Hs and not due to the marker fusion protein.

Screening of Phage Display Library and Identification of Anti-Caspase-3sdAbs.

A naïve llama V_(H)H phage display library was screened againstcaspase-3. Following three rounds of panning, 41/92 sdAbs clonesscreened by phage ELISA were positive for binding to caspase-3. Twentyfour of these were sequenced, giving two different sdAb sequences,namely, Casp1 and Casp2, occurring at frequencies of 22 and 2,respectively (FIG. 17). As shown in FIG. 16, both sdAbs are specific tocaspase-3 and do not bind to a control antigen. Both sdAbs weresubcloned as fusion proteins with C-terminal c-Myc-His₅ tag (SEQ ID NO:9), expressed in E. coli and purified to homogeneity for subsequentfunctional studies.

Additionally, both sdAbs were cloned into mammalian expression vectorspEGFP-N1 and pDsRed-N1 for in vivo intrabody functional studies.

Increasing the Efficacy of sdAb Intrabodies by Converting them toPentabodies

In addition to stability and expression level, the efficacy ofintrabodies is also determined by affinity. Since the active forms ofBax and caspase-3 are multimers, their binding to sdAbs can be increasedby multimerizing their sdAb binding partners (i.e., increasing affinitythrough avidity increase). Converting sdAb monomers to pentabodies hasbeen shown to increase their apparent affinity by several thousandfoldfold without compromising their expression yields and stability(Zhang, J. et al., 2004a).

All six anti-Bax V_(H)Hs (FIG. 1) were pentamerized substantially by themethod of (Zhang, J. et al., 2004b) as described. All were shown to haveacquired drastic increase in binding affinity by surface plasmonresonance as shown previously (Zhang, J. et al., 2004c).

Panning and phage ELISA. A llama V_(H)H phage display library describedpreviously was used in panning experiments (Tanha, J. et al., 2002c).Panning against recombinant Bax and caspase-3 proteins was performed asdescribed (Tanha, J. et al., 2002b) except that, in the case of Bax inthe second and the third rounds, the phage elution additionally involvedMgCl₂/HCl treatment. First, the bound phages in the microtiter wellswere eluted with 200 μl TEA and neutralized with 100 μl 1 M Tris-HCl pH7.4 (Tanha, J. et al., 2002a). Then, the emptied wells were subsequentlyincubated with 100 μl of 4 M MgCl₂ at room temperature for 15 min. Theeluted phage was removed and the wells were incubated with 100 μl of 100mM HCl for five min at room temperature. The MgCl₂/HCl-eluted phageswere pooled, neutralized with 1.5 ml of 1 M Tris-HCl pH 7.4 and combinedwith the TEA-eluted phages. One ml of the combined phages was used toinfect E. coli for overnight phage amplification and the remaining 1 mlwas stored at −80° C. for future reference. V_(H)H clones wereidentified from the titer plates by plaque PCR and sequencing asdescribed (Tanha, J. et al., 2003). Following panning, phage clones fromtiter plates were amplified in microtiter wells (Tanha, J. et al., 2003)and screened for binding to Bax protein by standard ELISA proceduresusing a HRP/anti-M13 monoclonal antibody conjugate (AmershamBiosciences, Baie d'Urfe, QC, Canada) as the detection reagent.

Protein expression and purification. V_(H)Hs were cloned from the phagevector into the expression vectors by standard cloning techniques(Sambrook, J. Fritsch E. F. and Maniatis T, 1989). E. coli expression ofV_(H)Hs and subsequent purification by immobilized metal affinitychromatography were performed as described (Tanha, J. et al., 2003).Protein concentrations were determined by A₂₈₀ measurements using molarabsorption coefficients calculated for each protein (Pace, C. N. et al.,1995). Mammalian expression of V_(H)H fusion constructs was initiated byinserting the V_(H)H genes in the Hind III/BamH I sites of pEGFP-N1(V_(H)H-GFP fusion) or pDsRed1-N1 (V_(H)H-RFP fusion) (BD Biosciences,Mississauga, ON, Canada) (FIG. 3A). The V_(H)H recombinant vectors weresubsequently used to transfect human neuroblastoma cells (SHSY-5Y) asdescribed below.

V_(H)H pentabody constructions. Pentabody cloning, expression,purification and binding analysis by surface plasmon resonance werecarried out as described (Zhang, J. et al., 2004d)

Surface plasmon resonance. Equilibrium dissociation constant, K_(D), forthe binding of Bax2 V_(H)H to Bax was derived from SPR data collectedwith BIACORE 3000 biosensor system (Biacore Inc., Piscataway, N.J.). Tomeasure the binding, 1800 RUs of protein Bax or 1100 RUs of a referenceFab were immobilized on research grade CM5 sensor chips (Biacore Inc.).Immobilizations were carried out at concentrations of 12 □g/ml (Bax) inpH 4.0 or 25 □g/ml (Fab) in pH 4.5, 10 mM sodium acetate buffer, usingthe amine coupling kit provided by the manufacturer. Analyses werecarried out at 25° C. in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3mM EDTA and 0.005% P20 surfactant at a flow rate of 40 □l/min, andsurfaces were regenerated by washing with the running buffer. Data werefit to a 1:1 interaction model simultaneously using BIAevaluation 4.1software (Biacore Inc.) and K_(D) was subsequently determined.

Cell culture. Human neuroblastoma (SHSY-5Y) cells (ATCC, Manassas, Va.)were grown in complete medium consisting of DMEM Ham's F12 media(Invitrogen Canada Inc., Burlington, ON, Canada) with the addition of 2mM L-glutamine (Invitrogen Canada Inc.) and 10% (v/v) fetal bovine serum(Sigma, Oakville, ON, Canada) and 20 μg/ml gentamycin (Invitrogen CanadaInc.). 200 μg/ml Geneticin (G418) (Invitrogen Canada Inc.) was added toall transfected cells. The cells were incubated at 37° C. with 5% CO₂and 95% humidity.

Statistical analysis. p values for all graphs were calculated usingStatistica Application for Windows 95, where p values less than 0.05were assumed to be statistically different.

V_(H)H isolation and mitochondria ROS measurement. SHSY-5Y cells weregrown to 70% confluence in 10-ml Petri dishes. The intact mitochondriawere isolated from these cells using a previously published method (Li,N. et al., 2003a). Mitochondria were suspended in solution containing0.25 M sucrose, 1 mM MgCl₂, 10 mM HEPES, 4 mg/ml p-hydroxyphenyl aceticacid (PHPA) and 20 mM succinate (Sigma Canada). Mitochondrial ROSgeneration is measured by H₂O₂ generation rate, determinedfluorimetrically by measurement of the oxidation of PHPA coupled to thereduction of H₂O₂ by horseradish peroxidase (Sigma Canada), based on apreviously published protocol (Li, N. et al., 2003b).

Detection of cytochrome c release by Western blot. Cytochrome c releasewas detected after incubating isolated mitochondria with V_(H)H for 15min followed by exposure to Bax for 5 min, in solution containing 0.25 Msucrose, 1 mM MgCl₂, 10 mM HEPES, and 20 mM succinate. Samples were thenspun down at 10,000 g for 5 min separating proteins of whole intactmitochondrial (pellet) and those released due to mitochondrial membranepermealization (supernatant). Pellet and supernatant fractions weresolubilized in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) loading buffer and proteins (50 mg protein /well) werethen subjected to a 12% SDS-PAGE, followed by transfer on nitrocellulosemembrane. The blots were probed with monoclonal anti-cytochrome cantibodies (Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) followedby washing and a second incubation with horse radishperoxidase-conjugated anti-mouse antibodies. The blots were developedusing a ChemiGlow West kit (Alpha Innotech Corporation, San Leonardo,Calif.) and recorded using an Alpha Innotech Corporation Imaging System.Integrated density values were calculated using Chemilmanager V5.5program for Windows 95.

Mammalian cell transfection. Mammalian transfection of V_(H)H fusionconstructs was initiated by inserting the V_(H)H genes in the HindIII/BamH I sites of pEGFP-N1 (V_(H)H-green fluorescent protein (GFP)fusion), pDsRed1-N1 (V_(H)H-red fluorescent protein (RFP) fusion) orHind III/Not I site of pEGFP-N1 (V_(H)H) (BD Biosciences, Mississauga,ON, Canada). The V_(H)H recombinant vectors were propagated in E. coliand were purified using QIAprep® Spin Miniprep kit according to themanufacturer's instructions (QIAGEN, Mississauga, ON, Canada). Thepurified plasmids were subsequently used to transfect SHSY-5Y cellsusing Fugene 6 Transfection Reagent (Hoffmann-La Roche Ltd.,Mississauga, ON, Canada) following manufacturer's protocol. Forty eighthours after transfection, cells were transferred to complete DMEM media(as described above) containing 300 μg/ml Geneticin for selection ofpositive transfected cells for 1-2 weeks. Stable cell lines weresubsequently maintained in complete DMEM media as described above with200 μg/ml Geneticin.

Detection of V_(H)H expression in mammalian cells by Western blot. Equalamounts of protein extract (50 μg) from control cells containing GFPonly and cells expressing specific GFP linked anti-Bax V_(H)Hs wereresolved by SDS-PAGE and transferred to a nitrocellulose membrane. Theblots were probed with monoclonal anti-GFP antibodies (Sigma, SaintLouis, Mo.) after which they were washed and incubated with horse radishperoxidase-conjugated anti-mouse antibodies. The blots were developed asdescribed above.

Induction of oxidative stress. Working solutions of H₂O₂ was made bydiluting a 10 M stock of H₂O₂ solution with distilled water to aconcentration of 100 mM. SHSY-5Y cells were grown to approximately 70%confluence. Oxidative stress was induced by incubating the cells incomplete media containing either 100 μM or 200 μM H₂O₂ for 1 h or 3 h at37° C. The media was then replaced with fresh, complete media (withoutH₂O₂) and the cells were incubated for different time periods to monitorapoptotic features and oxidative stress parameters.

Monitoring nuclear morphology. Nuclear morphology was monitored as anindicator for apoptosis in cells by staining cells with Hoechst 33342(Invitrogen Canada Inc.) to a final concentration of 10 μM. Afterincubating for 10 min at 37° C., the cells were then examined under afluorescence microscope (Zeiss Axioskope 2 Mot plus, Gottingen, Germany)and fluorescence pictures were taken using a camera (QImaging,Gottingen, Germany). The images were processed using Improvision OpenLabv3.1.2, Jasc Paint Shop Pro v8.00 and Adobe Photoshop v8.0.

Mitochondrial membrane potential detection and measurement.Mitochondrial membrane potential was detected using JC-1 mitochondrialspecific dye (Invitrogen Canada Inc.). The cells were treated with 10 μMJC-1 and incubated for 40 min at 37° C. The cells were observed underthe fluorescent microscope and fluorescence pictures were taken andprocessed as described above. Alternatively, mitochondrial membranepotential stability was also quantified using Dual Sensor: MitoCasp™Assay (Cell Technology Inc, Mountain View, Calif.) as per manufacturer'sinstructions.

Monitoring plasma membrane flipping. Annexin V (Invitrogen Canada Inc.)was used to monitor plasma membrane flipping in cells according tomanufacturer instructions. After incubating for 15 min at 37° C., thecells were examined under the fluorescence microscope and fluorescencepictures were taken and processed as described above.

Lipid peroxidation determination. Lipid peroxidation in cells wasdetermined using the thiobarbituric acid-reactive substances (TBARS)reaction with malondialdehyde and related compounds as previouslydescribed (Sunderman, F. W. JrTakeyama N. et al., 1985-52002).

Caspase 3/7 activation measurement. The activation of Caspase 3/7 wasmeasured in cells using Apo 3/7 HTS™ High Throughput Screen Assay kit(Cell Technology Inc, Mountain View, Calif.) as per manufacturer'sinstructions.

Activation of Caspase 3 Via Induction of Oxidative Stress

Plated SHSY-5Y cells of approximately 70% confluency were treated with100 μM H2O2 (1 μL per mL of media) for 1 hour to produce reactive oxygenspecies (ROS). The ROS in turn cause permeability of the mitochondria,leaking cytochrome c and initiating the Caspase cascade, leading to theactivation of Caspase 3. After 1 hour the media was removed and replacedwith new media and the cells were incubated for 3 hours. Following theincubation, the plates were trypsinized (0.15% trypsin) to remove thecells from the plate and the samples were collected in tubes. The tubeswere centrifuged at 35000 rpm for 7 minutes at room temperature and thesupernatant was removed and the pellet was resuspended in 2-3mL of PBS.The suspension was centrifuged again at 35000 rpm for 7 minutes at roomtemperature, and once again the supernatant was removed. The pellet wasresuspended in a hypotonic buffer on ice for 10 minutes, homogenized,and centrifuged at 3000 rpm for 8 minutes at 4° C. The supernatant waskept as it contained the caspase 3 and a protein estimation wasperformed on it.

Caspase 3 Activity Assay

A fluorescence assay was used to evaluate the presence of active Caspase3. DEVD-AFC (MP-Biomedicals, Aurora, Ohio) was used as the fluorescentsubstrate. The substrate, in the presence of DEVD Buffer (0.1M Hepes, pH7.4, 2 mM DTT, 0.1% CHAPS, 1% sucrose) and active caspase 3 wasincubated at 37° C. for 60 minutes and fluorescence was measured at 400nm excitation and 505 nm emission using the spectra max Gemini XS(Molecular Devices, Sunnyvale, Calif.). Caspase 3 activity was measuredas relative to the level of fluorescence.

Treatment of Active Caspase 3 with sdAbs and Measurement of Fluorescence

Following the protocol set out above, the effects on activity of Caspase3 could be monitored upon treatment with the sdAbs. The sdAbs were addedso that concentration would be equal to that of the isolated caspase 3and decreases in fluorescence could be monitored as the decrease inactivity of Caspase 3. The sdAbs were incubated with the active caspase3 for 30 minutes at 37° C. prior to the addition of the DEVD-AFC bufferand substrate. Following incubation, the caspase and sdAbs were added toDEVD buffer in a 96 well plate and the substrate was added. This wasincubated for 60 minutes and fluorescence was read.

Each experiment was performed in triplicate as described above and thiswas done four times. The control was active caspase 3+DEVDbuffer+DEVD-AFC, with caspase 3 single domain antibody 1 decreasingcaspase 3 activity and single domain antibody 2 increasing caspase 3activity (FIG. 18).

It is understood that the examples described above in no way serve tolimit the true scope of this invention, but rather are presented forillustrative purposes.

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Tanha, J., Muruganandam, A., and Stanimirovic, D. (2003). Phage DisplayTechnology for Identifying Specific Antigens on Brain Endothelial Cells.Methods Mol. Med. 89: 435-450.

Yau, K. Y., Dubuc, G., Li, S., Hirama, T., MacKenzie, C. R., Jermutus,L., Hall, J. C., and Tanha, J. (2005). Affinity maturation of a V(H)H bymutational hotspot randomization. J Immunol Methods 297: 213-224.

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Selection by phage display of llama conventional V(H) fragments withheavy chain antibody V(H)H properties. J. Immunol. Methods 263: 97-109.

Tanha, J., Muruganandam, A., and Stanimirovic, D. (2003). Phage DisplayTechnology for Identifying Specific Antigens on Brain Endothelial Cells.Methods Mol. Med. 89: 435-450.

Yau, K. Y., Dubuc, G., Li, S., Hirama, T., MacKenzie, C. R., Jermutus,L., Hall, J. C., and Tanha, J. (2005). Affinity maturation of a V(H)H bymutational hotspot randomization. J Immunol Methods 297: 213-224.

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We claim:
 1. An isolated single-domain antibody having an amino acidsequence that comprises at least one of SEQ. ID NO: 7 or SEQ. ID NO: 8.2. An isolated single-domain antibody having a binding affinity forcaspase-3 and having an amino acid sequence encoded by a nucleic acidsequence that comprises at least one of SEQ ID NO: 16 or SEQ ID NO:17.3. A single-domain antibody as claimed in claim 1 that is fused to apolypeptide.
 4. A single-domain antibody as claimed in claim 1 that isfused to an antibody domain.
 5. A single-domain antibody as claimed inclaim 4 wherein the antibody domain is an Immunoglobulin (Ig) lightchain variable domain (VL), an Ig heavy chain variable domain (VH), acamelid (camels and llamas) heavy chain antibody variable domain (VHH),a nurse shark and wobbegong shark Ig new antigen receptor(IgNAR)variable domain (VH), or a T cell receptor variable domain.
 6. Asingle-domain antibody as claimed in claim 3 wherein the polypeptide isa marker protein.
 7. A single-domain antibody as claimed in claim 3wherein the polypeptide is a signal protein or sequence determiningcellular localization.
 8. A single-domain antibody as claimed in claim 7wherein the signal protein is a mitochondrial transport signal protein.9. A single-domain antibody as claimed in claim 3 wherein thepolypeptide has the ability to cross cell membranes.
 10. A single-domainantibody as claimed in claim 9 wherein the polypeptide is a membranetranslocating sequence or protein transduction domain.
 11. Asingle-domain antibody as claimed in claim 9 wherein the polypeptide isan internalizing protein.
 12. An immunoliposome comprising asingle-domain antibody as claimed in claim
 1. 13. A multimer comprisingat least two single-domain antibodies as claimed in claim
 1. 14. Amultimer as claimed in claim 13 comprising five single-domainantibodies.
 15. A method for modulating the symptoms of apoptosis in acell and/or modulating the effects of oxidative stress in a cell, invitro or ex vivo, the method comprising the steps of exposing the cellto at least one single domain antibody that comprises at least one ofSEQ ID NO: 7 or SEQ ID NO: 8 and allowing binding of the at least onesingle-domain antibody to caspase-3.
 16. A method as claimed in claim 15further comprising the steps of delivering a nucleic acid sequence whichencodes the at least one single-domain antibody into the cell andallowing expression of the at least one single-domain antibody in thecell.
 17. A method as claimed in claim 16 further comprising the stepsof fusing the single-domain antibody to a polypeptide which permitsmembrane translocation or internalization of the single-domain antibodyinto the cell.
 18. A method for identifying a second single-domainantibody having a binding affinity for an apoptotic protein, said methodcomprising the steps of: a) providing a first single-domain antibody asdefined in claim 1; b) constructing an antibody library based on astructure of the first single domain antibody; and c) screening theantibody library for single-domain antibodies having a binding affinityfor the apoptotic protein.